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Oxidative stress induced paclitaxel-derived carbon dots inhibit glioblastoma proliferation and EMT process

Journal of Nanobiotechnology volume 23, Article number: 310 (2025) Cite this article

Abstract

Glioblastoma represents the most prevalent and deadly form of brain tumor with limited therapeutic drugs. The existence of the blood-brain barrier (BBB) hinders drugs permeate to the brain efficiently. Nowadays, nano-formulations, particularly carbon dots, have emerged as promising candidates for targeting and treating brain diseases. In this study, we report the synthesis of a novel carbon dots, PTX-CDs, using a one-step hydrothermal method with paclitaxel (PTX) as the precursor. PTX-CDs shows increased water solubility by about 1000 times in comparison with PTX. Moreover, PTX-CDs effectively penetrates the BBB and exerts significant anticancer effects. In detail, PTX-CDs accumulates in mitochondria of tumor cells without adding extra targeted molecules, resulting in the damage of mitochondrial membrane potential and increased reactive oxygen species (ROS) level. Transcriptome profiling revealed that PTX-CDs disturbs the cell-cycle by inducing arrest at the G2/M phase, thereby inhibiting cell proliferation. PTX-CDs further decreased cell invasion by inhibiting the epithelial-mesenchymal transition (EMT) process in glioblastoma cells. PTX-CDs significantly inhibited the growth of intracranial tumors in orthotopic glioblastoma mice model and prolonged the survival of tumor-bearing mice. This study presents a viable strategy to develop CDs-based therapeutic agent for glioblastoma using the conventional chemotherapeutic drugs.

Introduction

Glioblastoma, with an incidence rate of approximately 6.4 per 100,000 individuals, stands as the most aggressive form of brain tumor. It accounts for 57.3% of all malignant brain neoplasms [1]. The prognosis of glioblastoma remains poor despite of comprehensive treatments. The median survival of patients with glioblastoma is only 14 months after standard care of surgical resection, radiotherapy, and chemotherapy. The survival is extended by 6 months even when tumor-treating fields therapy is performed for glioblastoma patients [2, 3]. It is almost impossible for complete ablation of glioblastoma by surgical resection due to its high invasiveness. Additionally, high heterogeneity and complex tumor microenvironment of glioblastoma further impedes the therapeutic efficiency of radiotherapy, tumor-treating field therapy [4,5,6]. The drugs available for clinical treatment of glioblastoma are very limited, as the existence of the blood brain barrier (BBB) hinders drugs permeate to the brain. Over the past years, only 4 chemotherapeutic agents had been approved by FDA for the clinical treatment of glioblastoma, including temozolomide (TMZ), lomustine, carmustine and bevacizumab. However, long-term use of these reagents can lead to decreased sensitivity and even chemo-resistance [7]. 90% of glioblastoma patients recurred after current therapies. Therefore, it is urgent to develop new drugs that can efficiently bypass BBB for glioblastoma treatment.

Nanoparticles has been used in targeted drug delivery, genetic engineering, detection of biomarkers, diagnostics and many other uses. Rapid development of nanomedicine has brought new opportunities for the treatment of brain glioblastoma. Nanomedicine exhibits better cell-specific toxicity against tumors, chemotherapeutic sensitization, inhibition of angiogenesis, and induction of tumor starvation, among other tumor therapeutic benefits. Moreover, nanoparticles engineered with ligands conjugation for brain targeting, membrane coating for brain targeting, enabled them to efficiently cross the BBB [8, 9]. To use nanoparticles for the treatment of glioblastoma, the efficacy and safety of them should be considered and weighed [10]. The gathering of amount number of nanoparticles in the brain may induce some serious mental diseases, so the intracranial nanomedicines must have high biological safety and degradability.

Carbon dots, ranging in size from 1 to 100 nm, have garnered significant attention owing to their excellent biocompatibility, diverse physicochemical characteristics, cost-effectiveness, and high stability [11]. These versatile carbon-based nanoparticles can be categorized into carbon quantum dots, graphene quantum dots, carbon nanodots, polymer dots, and carbon-copolymerized dots [12]. Carbon dots have been engineered for tumor therapy by triggering apoptosis, autophagy, or ferroptosis [13,14,15,16,17,18]. Notably, carbon dots have shown considerable promise in the treatment of glioblastoma. Muhammad et al. developed carbon dots with natural plant leaves which effectively induced autophagy and apoptosis in drug-resistant glioblastoma cells based on their nano-enzymatic activities [19]. Carbon dots synthesized by metformin and gallic acid can freely penetrate the BBB and induce glioblastoma cells ferroptosis [20]. Furthermore, carbon dots were reported to be used as effective sonosensitizers for sonodynamic therapy of glioblastoma [21]. These studies indicated carbon dots as promising candidates for glioblastoma treatment. Due to the properties of carbon dots and retention effect, the carbon dots agent will have BBB permeability and tumor cell targeting [22]. Encapsulating the clinical approved chemotherapy drugs into carbon dots that enhance their BBB permeability and clinical availability may represent a promising strategy for glioblastoma therapeutics.

Paclitaxel (PTX) is a chemotherapeutic agent renowned for its ability to inhibit tubulin depolymerization and exert potent anti-cancer effects. However, the poor water solubility of PTX has limited its clinical application. Conventional formulations using Cremophor EL and ethanol to dissolve PTX leads to significant toxic side effects. Nano-formulations of PTX, such as albumin-bound PTX, polymeric micellar PTX, polymer-PTX couples, and liposome-encapsulated PTX significantly reduce the toxicity of PTX and greatly improve its anti-tumor efficiency [23]. For gliomas, PTX is probably 1400 times more potent than TMZ in glioma cell lines [24]. However, PTX showed low efficacy in phase 2 clinical trials when systemically administered for patient with recurrent malignant gliomas [25], as PTX does not cross the blood brain barrier [26]. Although albumin-bound PTX was markedly more permeable than PTX in brain tissue that had undergone BBB disruption [27], it necessitates modification to enhance its BBB permeability and therapeutic impact.

In this study, we synthesized a new CDs (PTX-CDs) by hydrothermal method using PTX. The solubility of PTX-CDs was significantly improved by probably 1000 times higher than PTX. The material characteristics of PTX-CDs was investigated, including particle size, Fourier-transform infrared (FTIR) spectrum, element composition, surface active group and zeta potential. The biological characteristics of PTX-CDs have also been verified. Preferable penetration efficiency of PTX-CDs was observed and PTX-CDs induced glioblastoma cells apoptosis by exerting on mitochondria to trigger reactive oxygen species (ROS) production. We also reported that PTX-CDs inhibited cell proliferation by inducing cell cycle arrest at the G2/M phase. PTX-CDs significantly suppressed the epithelial-mesenchymal transition (EMT) process which may prevent tumor invasion and metastasis (Scheme 1). Moreover, glioblastoma bearing mice treated with PTX-CDs showed reduced tumor volume and prolonged survival. This study highlights a promising candidate for therapeutic application against glioblastoma and a hydrothermal synthesis approach for CDs using chemotherapeutic agents.

Materials and methods

Synthesis of PTX-CDs

The synthesis of PTX-CDs was achieved via a straight forward one-pot hydrothermal method. Initially, 30 mg of PTX was dispersed in 5 ml ethanol followed by adding 25 ml deionized water. The mixture was sonicated to keep homogeneous dispersion. The solution was then transferred to a Teflon-lined autoclave, which was subsequently sealed within a stainless steel reactor housing. The assembly was heated in an oven at a temperature of 150 °C for a duration of 8 h. Upon cooling to ambient temperature, the suspension was centrifuged and filtered to eliminate any insoluble materials, yielding a clear brown solution. This solution was further purified by dialysis against water using a 1000 Da membrane. The purified solution of PTX-CDs was subsequently stored at -80 °C for further experiments.

Characterization of PTX-CDs

Transmission electron microscopy (TEM) images of PTX-CDs were obtained using a HT7800 microscope (Hitachi). FTIR spectra were recorded with a Nicoletâ„¢ iS50 FTIR spectrometer (Thermo Fisher Scientific). X-ray photoelectron spectroscopy (XPS) data were acquired on an AXIS Ultra DLD spectrometer (Kratos Analytical). Energy Dispersive X-Ray (EDX) images of PTX-CDs were obtained by Regulus-8100 (Hitachi). Fluorescence spectra and ultraviolet-visible (UV-Vis) absorption spectra were measured using an F97 Pro fluorescence spectrophotometer and an Agilent Cary 300 Scan UV-Vis spectrophotometer, respectively.

Cell culture

The mouse glioblastoma cell line GL261-Luci was generously provided by Dr. Minxuan Sun (SIBET, CAS). The human glioblastoma cell lines U251 and U87-MG, as well as mouse brain microvascular endothelial cells bEnd.3, were procured from Servicebio (Wuhan, China). The U251, U87-MG, and GL261-Luci cells were cultured in DMEM supplemented with 10% FBS and 1% of Penicillin/Streptomycin. bEnd.3 cells were maintained in RPMI 1640 medium supplemented with 10% FBS and 1% of Penicillin/Streptomycin. All cells were incubated at 37 °C in 5% CO2 humidified atmosphere incubator.

Cytotoxicity evaluation of PTX-CDs

Glioblastoma cells (5,000 per well) were seeded in a 96-well plate and cultured overnight. Subsequently, the cells were treated with various concentrations of PTX-CDs and TMZ (12.5, 25, 50, 100, and 200 µg/ml) for 48 h. Then the culture medium was replaced with fresh medium containing 10% WST-1 and incubated for an hour at 37 °C. The absorbance at 450 nm was measured using a microplate reader (Synergy H1, BioTek). The cell viability was calculated as follows: Cell Viability (%) =\(\:\:\frac{{\text{O}\text{D}}_{450}\:\text{a}\text{t}\:\text{d}\text{i}\text{f}\text{f}\text{e}\text{r}\text{e}\text{n}\text{t}\:\text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n}\text{s}}{{\text{O}\text{D}}_{450}\:\text{a}\text{t}\:0\:{\upmu\:}\text{g}/\text{m}\text{l}}\:\times\:\:100\:\text{\%}\). To quantify cell death, U251 cells were treated with different concentrations of PTX-CDs (0, 12.5, 50, 200 µg/ml) for 48 h, then stained with the Annexin V-FITC/PI Apoptosis Detection Kit and tested by flow cytometry. Flow cytometry data were analyzed with FlowJo software (FlowJo LLC, Ashland, OR, USA), and apoptotic stages were differentiated based on established criteria [16].

In vitro BBB model

bEnd.3 cells were seeded in Transwell inserts at 1 × 106 cells/well, and the culture medium was replaced every day. As the cells grow into a continuous and dense layer on the membrane, the PTX-CDs were added into the top chamber. After 4 h of incubation, the medium in the bottom chamber was collected for measuring the fluorescence intensity via microplate reader. The permeability of the BBB model was determined by quantifying the fluorescence intensity of PTX-CDs that penetrated to the bottom chamber. To explore the mechanism of PTX-CDs crossing the BBB, the endothelial cells on the inserts were pre-treated with 5 µg/ml chlorpromazine (CPZ), 50 µg/ml genistein, 5 µg/ml methyl-β-cyclodextrin (M-β-CD) or 10 µg/ml amiloride for 2 h, and then the permeability was measured following the same steps as described above.

Cellular uptake/release assay

To assess the uptake of PTX-CDs, U251 cells were seeded in cell culture plates and cultured overnight. The cells were then incubated with PTX-CDs at a concentration of 100 µg/ml for 10, 30, or 60 min. After that, cells were washed twice with phosphate-buffered saline (PBS) to remove extra PTX-CDs, and cellular images were captured using a laser scanning confocal microscope (Nikon). For the release study, U251 cells were treated with PTX-CDs (100 µg/ml) for 60 min, after which the supernatant was aspirated and replaced with fresh media for continued incubation. At 1 and 8 h post-treatment, cellular uptake and intracellular distribution of PTX-CDs were observed and imaged using the same confocal microscopy setup. For flow cytometry, cells were treated with 100 µg/ml PTX-CDs. Cells without PTX-CDs treatment were used as controls. After 8 h, the cells were trypsinized, resuspended in PBS containing 0.5% fetal bovine serum, and then analyzed by flow cytometry.

Cellular localization of PTX-CDs and mitochondrial status analysis

U251 cells were seeded in confocal dishes and cultured overnight. The cells were then incubated with PTX-CDs at the concentration of 100 µg/ml for 30 min. Following this incubation, organelles were stained with specific probe (Mito-Tracker Red CMXRos, Golgi-Tracker Red, ER-Tracker Red and Lyso-Tracker Red) according to the manufacturer’s protocol.

To evaluate mitochondrial damage post PTX-CDs treatment, cells were exposed to PTX-CDs at the concentration of 100 µg/ml for 24 h. Subsequently, Cells were fixed with 4% glutaraldehyde and used for TEM or mitochondria were stained with both Mito-Tracker Red CMXRos and JC-1, following the instructions provided by the manufacturer. All cellular imaging was performed using a laser scanning confocal microscope.

ROS detection

U251 cells were co-incubated with PTX-CDs at the concentration of 100 µg/ml for 6 h. Then the cells were incubated with 10 µM 2’,7’-Dichlorodihydro-fluorescein diacetate (DCFH-DA) for 30 min at 37 °C. Intracellular ROS level was assessed by fluorescence microscopy.

RNA-seq analysis

Total cellular RNA was extracted using Trizol reagent, and the integrity of the RNA was assessed through nucleic acid electrophoresis. For each sample, 1000 ng of RNA was reverse transcribed to cDNA, which served as the template for library construction using the KAPA Hyper Prep Kit (KAPA, KK8504). The size and quality of the constructed libraries were verified with a Fragment Analyzer 1.0.2.9, ensuring that the fragment distribution was predominantly between 300 and 700 base pairs (bp). The effective concentration of the libraries was precisely quantified by quantitative PCR (qPCR), with the requirement that the effective concentration be greater than 10 nmol/L to guarantee library quality. Sequencing was conducted on an Illumina NovaSeq 6000 platform, employing a paired-end (PE150) strategy. Differential gene expression analysis was performed using the DESeq2 package, with thresholds set at|log2 Fold Change| > 1 and p < 0.05 to identify significantly differentially expressed genes (DEGs). The RNA sequencing and associated data analysis were carried out by Geek Gene Technology Co., Ltd. (Beijing, China).

Cell proliferation assay

After treatment with 100 µg/ml PTX-CDs or a control regimen for 24 h, 2,000 glioblastoma cells per well were seeded in 96-well plate. The cell viability on various days was tested by WST-1 assay and the proliferation rate in each group was determined by the following formula: Cell proliferation =\(\:\:\frac{{\text{O}\text{D}}_{450\:}\text{o}\text{n}\:\text{d}\text{i}\text{f}\text{f}\text{e}\text{r}\text{e}\text{n}\text{t}\:\text{d}\text{a}\text{y}\text{s}}{{\text{O}\text{D}}_{450\:}\text{o}\text{n}\:\text{D}\text{a}\text{y}\:0}\). For the clone formation assay, 200 cells were seeded in each well of the 12-well plate. After two weeks of colony growth, the cells were fixed with 4% paraformaldehyde solution and stained with 0.1% crystal violet to visualize the colonies.

Cell cycle assay

Glioblastoma cells were treated with PTX-CDs at concentrations of 10, 25, and 50 µg/ml for 24 h. Then cells were collected, washed with PBS, and fixed in 70% ethanol overnight at -20 °C. Following fixation, the cells were centrifuged and washed with PBS, and then incubated with solution containing 50 µg/ml propidium iodide (PI) and 20 µg/ml RNase A for 30 min in the dark at 37 ℃. The cell cycle distribution was determined using flow cytometry. Data analysis was performed using FlowJo software.

Cell migration and invasion assays

For the Transwell migration assay, 104 glioblastoma cells treated with PTX-CDs were resuspended in 100 µl serum-free media and added into the chamber of the Transwell. Then the chamber was put into the lower well filled with 500 µl medium containing 10% FBS. After incubation for 48 h, the migrated cells were tested by fixation of 4% paraformaldehyde solution and staining with 0.1% crystal violet. Images of five representative fields were captured from each group, and the number of migrated cells was quantified. For the invasion assay, the membrane of the Transwell chamber was precoated with 1:8 dilution of Matrigel. And treated glioblastoma cells were then plated in the chamber as described above.

Western blotting

Cells were lysed in cold Cell Lysis Buffer for 30 min. The cell lysates were centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatants were collected. Protein concentrations were determined using the Detergent Compatible Bradford Protein Assay Kit. Equal amounts of protein samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes on ice. After blocking with 5% skim milk for 1 h at room temperature, the PVDF membranes were incubated with the respective primary antibodies overnight at 4 °C. Then incubation with horseradish peroxidase-conjugated secondary antibodies was performed at room temperature for 1 h. The blots were developed using the BeyoECL Star reagent and the resulting signals were documented using the ChemiScope 6300 Imaging System. All antibodies used in this assay are listed in Supplementary Tables 1 and were diluted to the concentrations recommended by the manufacturers.

In vivo anti-tumor assay

All animal experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Biological Research Ethics Committee of the Chinese Academy of Sciences (SIBET, CAS [2024-C001]). The 6–8 weeks old male C57BL/6J mice were maintained in a pathogen-free environment under a 12/12 h light-dark cycle. After one-week acclimatization, 1.5 × 105 GL261-Luci cells in 3 µl of PBS were stereotactically injected into the striatum of each mouse (coordinates: anterior 0.8 mm, lateral 2.0 mm, ventral 3.5 mm). Two weeks post-injection, bioluminescence imaging was performed using a Tanon ABL-X5 In Vivo Imaging System. Mice exhibiting a bioluminescence fluorescence intensity greater than 5 × 106 p/sec/cm2/sr were randomly divided into three groups. Mice were then injected by tail vein with TMZ (1 mg/ml, 200 µl), PTX-CDs (1 mg/ml, 200 µl), or saline (200 µl) every 2 days for 12 days. Mice were subjected to in vivo imaging at specific time points to monitor tumor growth.

Statistical analysis

Numerical data were presented as mean ± standard error of the mean (SEM). Statistical significance between the data treated and control groups was examined by the unpaired t test or one-way ANOVA using GraphPad Prism 8.0 (GraphPad Software, USA). A value of p < 0.05 was considered statistically significant.

Results

Development and characterization of PTX-CDs

PTX is one of the most widely used anti-tumor drugs. However, PTX has been limited in glioblastoma due to poor water solubility and BBB permeability. We try to synthesize a new CDs agent via one-pot hydrothermal method based on PTX (PTX-CDs). TEM analysis revealed that the synthesized PTX-CDs possessed a uniform size distribution and excellent dispersion (Fig. 1A). The particle size distribution, analyzed from the TEM image, exhibited an average particle diameter of 20.09 nm (Fig. 1B). The UV-vis absorption spectra of PTX displayed two distinct peaks at 240 and 275 nm (Fig. 1C), which are assignable to the π→π* transitions of the C = O and C = C bonds, respectively [14]. In contrast, the presence of a broad peak at 210–350 nm for PTX-CD implied a simplification of the PTX fine structure.

Fig. 1
figure 1

Characterization of PTX-CDs. (A) The TEM image of newly synthesized PTX-CDs. (B) The size distribution of PTX-CDs based on counting of 215 particles. Scale Bar = 200 nm. (C) UV–vis spectra of PTX-CDs. (D) Photoluminescence (PL) spectra of PTX-CDs in aqueous solutions. Inset shows a photograph of PTX-CDs in aqueous solution under UV light (365 nm). (E) FT-IR spectrum of PTX-CDs. (F) XPS spectra of PTX-CDs

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The optical properties of PTX-CDs were further characterized by fluorescence spectroscopy. Photoluminescence measurements showed that maximum excitation wavelength PTX-CDs at 390 nm and maximum emission wavelength at 490 nm (Fig. 1D). PTX-CDs emitted blue-green fluorescence under irradiation with 365 nm UV lamp (Fig. 1D inset). The emission spectra varied in wavelength and intensity with different excitation wavelengths, highlighting the excitation-dependent fluorescence of PTX-CDs (Fig. S1A).

FTIR spectroscopy was employed to elucidate the chemical structure of PTX and PTX-CDs (Fig. 1E). The peaks at approximately 3334 cm− 1 and 2930 cm− 1 corresponded to the O-H/N-H and C-H stretching vibrations, respectively. The C-O stretching vibrations was responsible for the characteristic absorption at 1044 cm− 1. The absorption at 1524 cm− 1 was indicative of a C = C skeletal vibration [13, 18, 28]. These shared peaks indicated that PTX-CDs partially retained the structure of PTX, whereas the absence of peak at 880 cm− 1 and the stronger C = O absorption peak at 1705 cm− 1 of PTX-CDs suggested that the absent of aromatic structure of PTX as well as the exposed aldehyde group during the synthesis of PTX-CDs [29].

The elemental composition of PTX-CDs was analyzed by XPS and EDX (Fig. 1F and Fig. S2). In In the XPS spectra, the characteristic peaks at 284.8 eV, 399.6 eV, and 532.3 eV represented C 1s, N 1s, and O 1s respectively, with atomic percentages of 81.62% (C), 16.39% (O), and 1.99% (N). The high-resolution XPS spectrum of C 1s was deconvolved into three peaks at 284.8 eV, 286.2 eV, and 288.8 eV, corresponding to C-C/C = C, C-O, and C = O bonds (Fig. S1B). The high-resolution O 1s spectrum revealed two distinct peaks representing C-O at 532.2 eV and C = O at 533.3 eV (Fig. S1C) [13, 16, 28]. The low intensity of the N 1s peak, due to the low nitrogen content, was shown in Fig. S1D. Similar carbon/nitrogen atom percentage was shown by EDX measurement. These data collectively indicated that the synthesized PTX-CDs retained a portion of the chemical structure of the PTX precursor.

It was reported that the water solubility of PTX is less than 1 µg/ml [30]. We next tested the water solubility of the newly synthesized PTX-CDs and compared it with PTX. The same mass of PTX or PTX-CDs was resuspended in the same volume of water. PTX-CDs was completely dissolved under the concentration of 1.25 mg/ml, while PTX was noticeably insoluble at the same concentration. Then a small amount insoluble particles of PTX-CDs was seen under the concentrations of 2.5 mg/ml and 5 mg/ml (Figs. S3A-B). To further quantify the solubility of PTX-CDs, the UV absorption of different concentrations of PTX-CDs was determined at 300 nm. When the concentration was less than 1.25 mg/ml, the absorbance of PTX-CDs was positively correlated with its concentration. The absorbance of PTX-CDs kept all most the same when the concentration was increased to 1.5 or 2.0 mg/ml (Fig.S3C). So, the solubility was increased by more than 1000 times when PTX was synthesized into PTX-CDs. Then the anti-tumor and BBB penetration ability of PTX-CDs was further investigated to determine whether it could be used for glioblastoma treatment.

Uptake and therapeutic effects of PTX-CDs in glioblastoma cells

Previous studies had shown that carbon dots could be taken up in an ATP-dependent manner and targeted to different organelles [31]. So, we next detected the uptake of PTX-CDs by cells. Given the photoluminescent properties of PTX-CDs, we utilized confocal microscopy to investigate their cellular uptake by glioblastoma cells. As depicted in Fig. 2A, PTX-CDs were internalized within 30 min, and the fluorescent signal could persist for over 8 h post-incubation, indicating a robust and sustained presence within the cells. In addition, we used flow cytometry to detect the uptake of PTX-CDs by cells. As shown in the Fig. S4, a distinct green fluorescence signal was detected after PTX-CDs treatment, indicating that PTX-CDs was effectively taken up by the cells. The cytotoxic effects of PTX-CDs at various concentrations were assessed by performing WST-1 assays on U251, U87-MG, GL261 cell lines. And TMZ, which is a standard first-line chemotherapeutic agent for glioblastoma treatment, was used as positive control. Cell viability was significantly inhibited by both PTX-CDs and TMZ, while the inhibitory ability of PTX-CDs was much higher than TMZ (Figs. 2B-C and Fig. S5). Annexin V/PI staining and flow cytometry analysis showed that PTX-CDs induced cell apoptosis and the number of late stage apoptosis cells increased progressively with escalating concentrations of PTX-CDs (Fig. 2D). This finding indicated the dose-dependent cytotoxicity was induced by PTX-CDs on glioblastoma cells. As the potential of nanoparticles with smaller sizes to penetrate the BBB was reported [32], we then evaluated the permeability of PTX-CDs employed an in vitro BBB model based on its inherent photoluminescence. The permeability rate of PTX-CDs was calculated at approximately 19.8% (Fig. 2E). These results suggested that PTX-CDs could potentially be used in the treatment of glioblastoma. As PTX-CDs was not functionalized by the ligand, we speculated that the non-specific endocytosis of brain endothelial cells mediates its BBB penetration [33, 34]. Four different endocytosis inhibitors: chlorpromazine (CPZ), amiloride, genistein, and methyl-β-cyclodextrin (M-β-CD) Which are the inhibitors of clathrin-mediated endocytosis, macropinocytosis, caveolae-mediated endocytosis, and lipid raft-mediated pathways, respectively [35], were used in the BBB permeability assay. As shown in the Fig. S6, the permeability of PTX-CDs was significantly inhibited by amiloride, while slight inhibition was found after genistein treatment. Meanwhile, M-β-CD and CPZ showed almost no impact on the permeability of PTX-CDs. Thus, the penetration of PTX-CDs across the BBB was predominantly mediated by macropinocytosis in endothelial cells.

Fig. 2
figure 2

Uptake and anti-tumor effects of PTX-CDs in glioblastoma cells. (A) PTX-CDs (100 µg/ml) uptake by U251 cells from 30 min after treatment and released since 8 h after washing. Scale Bar = 100 μm. Cell viability of U251 (B) and U87-MG (C) treated with PTX-CDs and TMZ at different concentrations for 48 h. (D) Flow cytometry analysis showed PTX-CDs induced cell apoptosis which was positively correlated with concentration. (E) Penetration capability of PTX-CDs crossing the Transwell BBB model after incubation for 4 h

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PTX-CDs exerts on mitochondria and induces oxidative stress resulting in cell death

To elucidate the mechanism of cell death induced by PTX-CDs, we first investigated their subcellular localization. Given the positive Z-potential of PTX-CDs (Fig. S7), we hypothesized that they would be concentrated in the mitochondria following cellular uptake. To more accurately investigate the subcellular localization of PTX-CD, cells were co-treated with various organelle-specific probes and PTX-CDs, and the intracellular distribution of fluorescence was observed by confocal microscopy. As illustrated in Fig. S8, PTX-CDs was distributed throughout the cytoplasm. In addition to the mitochondrial localization, we found that PTX-CDs also exhibited significant accumulation in lysosomes, while their colocalization with the endoplasmic reticulum and Golgi apparatus was poor. Intact and tubular mitochondria was seen in untreated cells by Mito-Tracker staining, while diffuse and fragmented mitochondria was found in PTX-CDs treated cells (Fig. 3A). And a loss of mitochondrial membrane potential was seen by JC-1 staining in the PTX-CDs treated group while intact mitochondrial membrane potential was found in untreated cells (Fig. 3B). As shown in the Fig. 3C, treatment with PTX-CDs resulted in mitochondrial shrinkage and reduced number of mitochondrial cristae, underscoring the significant impact of PTX-CDs on mitochondrial morphology and function. As the damage of mitochondria may lead to the accumulation of intracellular ROS, we next tested the intracellular ROS level in PTX-CDs treated and untreated groups using ROS probe. Significant differences in ROS level were observed in these two groups (Fig. 3D). To investigate whether the cell death was leaded by PTX-CDs induced oxidative stress, the cells were treated with various antioxidants, including β-mercaptoethanol (BME), N-acetylcysteine (N-Ace), glutathione (GSH), and dithiothreitol (DTT). These antioxidants attenuated cell death in PTX-CDs treated group (Figs. 3E-H), further demonstrating that the PTX-CDs induced oxidative stress was the main cause of cell death. In summary, our data suggested that generation of ROS through PTX-CDs induced oxidative stress caused glioblastoma cell death, which was mediated by the damage of mitochondrial membrane potential.

Fig. 3
figure 3

PTX-CDs exerted on mitochondria and induced oxidative stress in glioblastoma cells. Changes in mitochondrial morphology (A) and membrane potential (B) after 24 h treatment of PTX-CDs. (C) TEM image of U251 cells upon PTX-CDs treatment for 24 h. (D) ROS production in glioblastoma cells incubated with or without PTX-CDs. (E-H) Various antioxidants rescued the cell viability decreased by PTX-CDs

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RNA-seq analysis revealed disruption of the cell cycle induced by PTX-CDs

To delineate the potential therapeutic mechanisms of PTX-CDs, RNA- sequencing was performed using untreated and PTX-CDs treated U251 cell lines. Three biological replicates were analyzed in each group. The data reliability was confirmed through principal component analysis (PCA) and t-distributed stochastic neighbor embedding (t-SNE), which revealed significant transcriptomic differences among the samples (Fig. S9). The global distribution of differentially expressed genes was depicted in Fig. 4A and B, with 828 genes up-regulated and 414 genes down-regulated (|log2 Fold Change| > 1, p < 0.05). Significant differences between untreated control and PTX-CDs treated groups in the transcriptome were illustrated and shown in the heatmap (Fig. 4C). Gene ontology (GO) annotation of the differentially expressed genes revealed significant enrichment of genes associated with cell cycle and DNA replication (marked by red boxes in Fig. 4D), suggesting that substantial disruption of the cell cycle was induced by PTX-CDs (Fig. 4D). These results indicated that PTX-CDs treatment may lead to cellular DNA damage and cell cycle arrest. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the differentially expressed genes were enriched in pathways related to cellular metabolism, including amino acid biosynthesis, AGE-RAGE signaling pathway, carbon metabolism, AMPK signaling pathway, FoxO signaling pathway, and PI3K-AKT signaling pathway, and so on (Fig. 4E). Besides, pathways related to DNA replication and cell cycle were also enriched. In summary, we found that a broad spectrum of transcriptional changes in glioblastoma cells were induced by PTX-CDs, especially in cell cycle and DNA replication related progress, which indicated the potential of PTX-CDs to be used for inhibiting tumor cell proliferation.

Fig. 4
figure 4

RNA-seq analysis revealed disruption of the cell cycle by PTX-CDs. (A) Volcano plots displayed the differentially expressed genes in U251 cells treated with or without PTX-CDs. Genes expressed without significant differences were indicated as black dots. (B) Statistical map of up and down expressed genes. (C) Heat map analysis of differently expression of genes after PTX-CDs treatment. (D) The GO analysis showed that the cell cycle related genes were interrupted by PTX-CDs. (E) KEGG analysis showed that DNA replication related pathway was mostly enriched after PTX-CDs treatment

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PTX-CDs inhibited cell proliferation by inducing cell cycle arrest in G2/M phase

As the RNA-sequencing data showed that cell cycles were disrupted by PTX-CDs, we next tested whether the cell proliferation would be inhibited by PTX-CDs. Colorimetric WST-1 assay was used to check the proliferation rate of glioblastoma cell lines U251 and GL261, treated with or without PTX-CDs. The results showed that the cell proliferation was inhibited by about 80% based on PTX-CDs treatment (Fig. 5A). Colony formation assay further confirmed that the colony-forming ability of glioblastoma cells was significantly inhibited by PTX-CDs in a dose-dependent way (Fig. 5B and Fig. S10). To elucidate the mechanism of PTX-CDs induced inhibitory effects on cell proliferation, PI staining was utilized to analyze the cell cycle distribution. As shown in Fig. 5C, decreased number of cells in the G1 phase and increased number of cells in the G2/M phase were seen in PTX-CDs treated group, compared with untreated control group. It suggested that a substantial proportion of cells were unable to progress effectively into the division phase after treatment with PTX-CDs. Western blot analysis of proteins critical for G2/M cycle regulation, such as CDC25C, Cyclin B1, and CDK1, revealed significant decreased expression level of those proteins after PTX-CDs treatment (Fig. 5D). These results suggested that PTX-CDs disrupt the normal cell cycle by leading to cell cycle arrest in the G2/M phase. In summary, our findings indicated that PTX-CDs inhibited cell proliferation by inducing cell cycle arresting at the G2/M phase, which suggested PTX-CDs as a potential therapeutic strategy for glioblastoma treatment by preventing cell division and proliferation.

Fig. 5
figure 5

PTX-CDs inhibited glioblastoma cell proliferation. (A) Proliferation of glioblastoma cell treated and untreated with PTX-CDs. (B) Colony forming ability of glioblastoma was inhibited by PTX-CDs. (C) Cell cycle of glioblastoma cells was distributed by different concentrations of PTX-CDs. (D) The expression of CDC25C, Cyclin B1 and CDK1 in glioblastoma cells was decreased after PTX-CDs treatment for 24 h

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PTX-CDs inhibited EMT in glioblastoma cells

Treatment failure is very common in invasive and diffuse glioblastoma. In this study, we observed that tumor invasion was inhibited by PTX-CDs treatment. Initially, morphological change from spindle to cobblestone appearance in U251 and GL261 was found in after PTX-CDs treatment. It indicated inhibition of transition from epithelial to mesenchymal cells and loss of cell polarity and migrating capacity (Fig. 6A) [14]. As seen in migration and invasion assays, fewer than 15% of cells successfully migrated across the membrane after PTX-CDs treatment and the number of migrating and invasion cells was significantly reduced by PTX-CDs (Fig. 6B). Scratch assays also verified the inhibitory effects of PTX-CDs on U251 cell migration (Fig. S11). PTX-CDs also induced morphological changes and inhibited their invasion and migration in the invasive U87-MG cells (Figs. S12-14). Additionally, the expression of EMT-related markers in U251 and GL261 cells treated with or without PTX-CDs was analyzed using western blot (WB). The expression of N-cadherin and Vimentin was decreased, while the expression of E-cadherin was increased after PTX-CDs treatment (Fig. 6C). Immunofluorescent staining confirmed decreased of Vimentin positive cells and increased of E-cadherin positive cells after PTX-CDs treatment in U251 cells, while the subcellular localization of Vimentin and E-cadherin was not altered (Fig. 6D). Quantitative real-time PCR (qPCR) assay further confirmed the inhibitory effects of PTX-CDs on EMT in U251 cells (Fig. S15). The expression of TGF-β was significantly inhibited by PTX-CDs after treatment for 12 h and 24 h. This further confirmed the inhibitory effect of PTX-CD on the EMT process (Fig. S16). All these findings suggested that EMT of glioblastoma cells could be effectively inhibited by PTX-CDs, which may further lead to decreased tumor cells invasion.

Fig. 6
figure 6

PTX-CDs inhibited EMT in glioblastoma cells. (A) Cell morphology of aggressive glioblastoma cells was affected by PTX-CDs. (B) Migration and invasion of glioblastoma cells were inhibited by PTX-CDs in Transwell assay. (C) Expression of N-cadherin and Vimentin in glioblastoma cells was decreased, while expression of E-cadherin was increased after PTX-CDs treatment for 24 h. (D) Immunofluorescent staining of E-cadherin and Vimentin in U251 cells after PTX-CDs treatment for 48 h

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Biosafety evaluation of PTX-CDs

The biosafety of nanomaterials is paramount to their translational potential. We conducted a preliminary assessment of the safety profile of PTX-CDs. L929 which is mouse fibroblasts cells was used as a non-cancer cell model to evaluate the toxicity of PTX-CDs on normal cells. L929 and U251 cells were treated with various concentrations of PTX-CDs respectively. As shown in Fig. S17, PTX-CDs was less toxic to L929 than that to U251 cells under the same concentration. Severe cytotoxicity was seen on both L929 and U251 cells under the concentration of 200 µg/mL and it indicated that PTX-CDs was considered safe within a certain concentration range, while high concentration of PTX-CDs exhibited toxicity to normal cells. Hemocompatibility was evaluated by co-incubating PTX-CDs with erythrocytes. And no significant hemolysis was observed even when the concentrations of PTX-CDs was up to 200 µg/ml, which suggested good hemocompatibility of PTX-CDs (Fig. S18). Systematic safety evaluation in vivo was further conducted by i.v. injection of PTX-CDs into healthy adult mice. No significant differences in body weight between the treated and untreated groups were seen throughout the whole treatment period (Fig. S19). In addition, mice tissues were obtained at different time points after intravenous injection to evaluate the tissue distribution. As shown in Fig. S20A, PTX-CDs was enriched in liver and kidney within 3 h, with a subsequent gradual decline in fluorescence intensity over time. This trend suggested that PTX-CDs were effectively cleared from the system via hepatic and renal excretory pathways. 48 h later, no obvious fluorescent signals were detected, indicating that the rapid plasma clearance rate of PTX-CDs [36]. Moreover, we used tumor-bearing mice to assess the brain permeability of PTX-CDs. As shown in Fig. S20B, PTX-CDs could effectively accumulate in the brain tissue. And due to the Enhanced Permeability and Retention (EPR) effect, their concentration was augmented specifically in the brains of mice with tumors. Thus, PTX-CDs exhibited an enhanced capacity to accumulate at tumor sites, thereby potentially mediating anti-tumor activities [37]. Histological examination of major organs (such as heart, liver, spleen, lung, kidney, and brain) via hematoxylin and eosin (H&E) staining revealed no significant organ damage attributable to PTX-CDs treatment (Fig. S21). Additionally, the liver, kidney, and myocardial function was assessed by measuring serum level of aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, creatinine (CREA), and creatine kinase (CK). All of the data value was within the normal range and negligible differences were seen between the two groups (Fig. S22). In summary, it was indicated that PTX-CDs was with favorable biocompatibility, which is a crucial prerequisite for their potential in in vivo anti-tumor applications.

Therapeutic effects of PTX-CDs for glioblastoma in vivo

As promising anti-tumor effects of PTX-CDs was seen in vitro, we next tried to evaluate the therapeutic efficacy of PTX-CDs in vivo. An orthotopic glioblastoma C57BL/6 mice model was established by injecting GL261-Luci cells into mice brain. 14 days later, intracranial tumorigenesis was detected by in Vivo Imaging System. Then tumor-bearing mice were randomly divided into 3 groups with 5 animals in each group, control (saline, 200 µL) and TMZ (1 mg/ml, 200 µl) and PTX-CDs (1 mg/ml, 200 µl). Each mouse was injected by the tail vein every 2 days for 12 days (Fig. 7A). As observed under an in Vivo Imaging System, mice treated with TMZ and PTX-CDs showed significant decrease in bioluminescence signal since day 20 compared with mice treated with saline (Fig. 7B). The relative bioluminescence quantification was shown in Fig. 7C. In addition, no mice were dead in the TMZ and PTX-CDs treated groups throughout the treatment periods while extensive deaths were seen in the control group. And the survival curve was shown Fig. 7D. Moreover, a significant decrease in body weight in control group was observed while steady body weight was kept in the TMZ and PTX-CDs groups (Fig. S23). Next, shrunk tumor tissue (H&E staining) and decreased Ki67 expression (immunohistochemistry staining) were found in PTX-CDs groups compared with the control (Figs. 7E-F). These results indicated the inhibitory ability of PTX-CDs in tumor growth in vivo.

Fig. 7
figure 7

PTX-CDs exhibited significant inhibitory effects on the tumor progression in orthotopic glioblastoma mice model. (A) Schematic illustration of PTX-CDs therapeutic schedule in orthotopic glioblastoma model. (B) Bioluminescence images of orthotopic GL261-Luc glioblastoma-bearing mice treated with saline, TMZ and PTX-CDs at day 14, 20, 26 post xenografting (Dosage: 1 mg/ml, 200 µl). (C) Quantified luminescence levels of bioluminescence images. (D) Survival curve of mice in each group with the indicated drug treatment. (E) H&E staining and (F) immunohistochemistry staining of Ki-67 on GBM tissues from tumor-bearing mice treated with PBS and PTX-CDs. The red curve represented the boundary between the tumor and normal brain tissue

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Discussion

Glioblastoma is a fatal primary brain cancer lacking effective therapeutic drugs. The BBB severely hinders the delivery of drugs into the central nervous system (CNS) from blood circulation system [20]. In recent years, nanoparticles, especially carbon dots (CDs), has been demonstrated promising applications for CNS diseases, especially in term of BBB penetration. Carbon dots developed using D-glucose and L-aspartic acid (CD-Asp) can freely penetrate the BBB and exhibit precise glioblastoma targeting capabilities [38]. Similarly, Carbon dots prepared by microwave assisted method using metformin and Citric acid as precursor (Met-CDs) successfully crossed the BBB and facilitated for living cell imaging [39]. These advancements highlight the expanding function of carbon dots. Moreover, carbon dots can serve as effective drug delivery vehicles to the brain. Carbon dots derived from 1,4,5,8-tetraminoanthraquinone and citric acid or L-tryptophan and D-glucose enabled specific imaging of brain tumors and facilitated treatment through the loading of chemotherapeutic agents such as topotecan hydrochloride or doxorubicin [22, 40]. Furthermore, coupling carbon dots with tumor-targeting ligands has been shown to enhance their targeting efficiency [41]. Carbon dots conjugated with glucose oxidase have also been explored for glioblastoma therapy [42]. Additionally, Deng et al. created carbon dots utilizing metformin and Gallic acid, which not only allowed cellular imaging but also demonstrated antitumor activity by inducing ferroptosis in glioblastoma cells [20]. However, most carbon dots are used for biological imaging of tumors or drug delivery, especially to the CNS. The antitumor efficacy of carbon dots often depended on the drug they carry or the ligands to which they are attached. In this study, we successfully synthesized novel carbon dots (PTX-CDs) using PTX as precursor via one-pot hydrothermal method, and demonstrated that the water solubility of PTX-CDs was significantly increased by more than 1000 times compared with PTX. PTX-CDs can freely penetrate the BBB, target tumor cells and accumulate in mitochondria. Most importantly, PTX-CDs exhibited significant antitumor effects independently, without necessitating additional modifications.

The BBB is a critical physical barrier to protect the brain from harmful substances, and it hinders most drugs permeate to the brain for the treatment. Typically, small lipophilic molecules can cross the BBB through passive diffusion, while larger molecules rely on carrier and receptor mediated transcytosis to overcome this barrier [33]. The capacity of carbon dots to cross the BBB can be attributed to their small particle size, as the tight junctions within the BBB possess gaps that permit particles smaller than 4 nm to pass [43]. Some carbon dots have been shown to cross the BBB via specific transport proteins [44], and conjugation with transferrin can facilitate entry into the CNS via receptor-mediated endocytosis [45]. The BBB crossing efficiency could also be enhanced through near-infrared irradiation [46]. Nanoparticles with different size cross the BBB via vary pathways [34]. It was reported that carbon dots synthesized with glucose may cross the BBB depending on glucose transporter proteins [44]. In our study, the permeability rate of PTX-CDs was tested at approximately 19.8%. And the average size of PTX-CDs was 20.09 nm, which is larger than the tight junction gaps and could not cross the BBB by passive diffusion. We hypothesized that PTX-CDs may cross the BBB via alternative mechanisms and the way that PTX-CDs crossed the BBB will be identified in our further study.

Here we showed that PTX-CDs was able to be taken up by glioblastoma cells and released after 8 h (Fig. 2A). PTX-CDs with positive charge underwent endocytosis and accumulate in the mitochondria with negative potential [47, 48]. Mitochondria, where the oxidative phosphorylation reactions occur, are always enriched with ROS. When mitochondria are damaged, the ROS released into the cytoplasm may trigger cell death, including apoptosis, necrosis, pyroptosis, and ferroptosis [49, 50]. Additionally, carbon dots can function as nanozymes, mimicking the structure and function of enzymes such as glutathione peroxidase, catalase, superoxide dismutase, and thiol peroxidase [18, 19], and leading to cellular oxidative stress and damage. Carbon dot-based nanozymes are considered to be an effective strategy for anti-tumor therapy. Our data displayed that PTX-CDs targeted mitochondria, induced oxidative stress by the damage of mitochondrial membrane potential and caused cell apoptosis in glioblastoma cells. RNA-seq analysis revealed the disruption of the cell cycle induced by PTX-CDs, resulting in cell cycle arrest. The cell cycle checkpoint transitions from the G1/S phase to the G2/M phase is regulated by CDK1-cyclinB1 [51]. In our study, the expression of CDK1-cyclinB1 and its upstream regulator CDC25C was significantly reduced by PTX-CDs treatment, which resulted in cell cycle arresting in the G2/M phase and limited cancer cell proliferation (Fig. 5).

Moreover, metastasis play an important role in tumor recurrence, and the activation of EMT can transform tumor cells into a highly invasive mesenchymal cells [16]. Inhibition of the EMT process is crucial for the treatment of aggressive glioblastoma. Here PTX-CDs were shown to inhibit the EMT process in types of glioblastoma cells, displaying their great potential in glioblastoma aggressive treatment (Fig. 6 and Figs. S11-16).

This study reported a strategy to synthesis carbon dots using chemotherapeutic drugs as precursor. The water solubility and the BBB penetration of the precursor was dramatically increased, while anti-tumor effects were well kept. This strategy suggested new direction to develop drugs for glioblastoma, which may greatly shorten the period of new medicine researches. The mechanism that PTX-CDs penetrate the BBB will be investigated which may provide great favor for further modification of PTX-CDs. And more chemotherapeutic drugs will be chosen and synthesized into carbon dots for glioblastoma treatment.

Conclusion

In summary, we synthesized a novel glioblastoma therapeutic carbon dots (PTX-CDs) using PTX which is clinical approved chemotherapeutic drug for many types of cancers. The solubility of PTX-CDs is highly increased compared with PTX. And PTX-CDs effectively penetrates the BBB, accumulates in tumor cell mitochondria, further induces cell death and decreased cell invasion by inhibiting the EMT in glioblastoma cells. PTX-CDs exerts significant anticancer effects with low biotoxicity in vivo, which demonstrates it as a potential intracranial tumor therapeutic agent. Particularly, we have reported a reliable strategy to encapsulate the conventional chemotherapeutic drugs into carbon dots that enhance their solubility and BBB permeability, while the anticancer effects were well kept. This work presents a promising strategy for development of CDs-based novel therapeutic agent for glioblastoma.

Scheme 1
scheme 1

Schematic illustration of the preparation of PTX-CDs and their multiple function for glioblastoma therapy

Full size image

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

This research was funded by the National Key R&D Program of China (Grand No. 2023YFB3210300), and the Youth Innovation Promotion Association of CAS (2022326).

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Authors and Affiliations

  1. School of Biomedical Engineering (Suzhou), Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230026, China

    Haiyang Yan, Huimin Miao, Jiukun Hu, Jinlin Pan, Jinyu Yao, Yuwei Du & Xinlu Li

  2. Department of Biomaterials and Stem Cells, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Science, Suzhou, 215163, China

    Haiyang Yan, Huimin Miao, Jiukun Hu, Jinlin Pan, Mingfeng Ge, Jinyu Yao, Yuwei Du, Xinlu Li, Li Li, Wen-fei Dong & Lixing Zhang

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Contributions

Haiyang Yan: Conceptualization, Data curation, Formal analysis, Writing – original draft, Writing – review and editing. Huimin Miao: Data curation, Methodology, Software. Jiukun Hu: Methodology, Resources. Jinlin Pan: Methodology, Visualization. Mingfeng Ge: Project administration, Supervision, Funding acquisition. Jinyu Yao: Methodology. Yuwei Du: Software. Xinlu Li: Visualization. Li Li: Funding acquisition, Supervision, Writing – review and editing. Wen-fei Dong: Funding acquisition, Supervision, Writing – review and editing. Lixing Zhang: Funding acquisition, Supervision, Writing – review and editing.

Corresponding authors

Correspondence to Li Li, Wen-fei Dong or Lixing Zhang.

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All animal experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Biological Research Ethics Committee of the Chinese Academy of Sciences (SIBET, CAS [2024-C001]).

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The authors declare no competing interests.

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Yan, H., Miao, H., Hu, J. et al. Oxidative stress induced paclitaxel-derived carbon dots inhibit glioblastoma proliferation and EMT process. J Nanobiotechnol 23, 310 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12951-025-03406-x

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12951-025-03406-x

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Journal of Nanobiotechnology

ISSN: 1477-3155

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